傅里叶变换红外光谱(FTIR)技术对滇龙胆组织培养的研究

野生药用植物资源的不断减少,使得寻找其原植物的合适替代品显得尤为重要。利用组培材料代替野生药用植物作为药源已取得重大进展,但利用傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FTIR)技术筛选合适的组培材料作为野生药用植物替代资源方面的应用鲜有报道。本研究采用FTIR结合偏最小二乘判别分析(partial least squares discriminant analysis,PLS-DA)对滇龙胆组织培养形成的愈伤组织(肉质部、茎、叶)、增殖苗(肉质部、茎、叶)、生根苗(根、茎、叶)进行比较。结果显示:(1)从原始FTIR光谱图上看,滇龙胆肉质部和根部峰形相似,茎和叶峰形相似;(2)二阶导数光谱图扩大了样品间的差异。在龙胆苦苷的主要吸收峰1612 cm-1附近,吸收峰强度依次为:生根苗叶增殖苗叶和生根苗茎增殖苗茎愈伤组织叶,愈伤组织茎及肉质部、增殖苗肉质部和生根苗根部在该处无吸收峰;(3)PLS-DA得分图表明,同一组培阶段相同组织部位样品聚集在一起,而愈伤组织、增殖苗、生根苗及其各组织部位能够较好的分开。其中:肉质部、根部与茎叶之间距离较远,表明其化学成分和含量可能差异较大;肉质部和根部样品间距离较近,茎和叶样品间距离也较近。二阶导数光谱图显示,组培材料有望代替其原植物满足药用需求;若以龙胆苦苷含量为评价对象,生根苗叶则可能具有更大的开发潜能,有望代替野生滇龙胆以缓解其资源稀缺局面。本研究结果表明,采用傅里叶变换红外光谱法可以简便有效地对药用植物不同组培阶段不同组织部位的替代潜力及开发利用进行初步评估。     

栽培条叶龙胆根中龙胆苦苷含量差异的研究

采用高效液相色谱法,测定栽培条叶龙胆根中龙胆苦苷的含量,结果表明:花末期(9月3日)龙胆苦苷含量高于果期(9月18日)和枯萎期(10月10日);同一根系中二龄根的含量显著高于一龄根;有性后代个体之间龙胆苦苷含量差异显著,对条叶龙胆进行高含量育种研究是可行的。     

滇龙胆不同部位裂环烯醚萜类物质含量比较分析

采用高效液相色谱法,对云南临沧云县滇龙胆不同部位龙胆苦苷、獐牙菜苦苷、当药苷含量进行分析.结果显示:(1)龙胆苦苷、獐牙菜苦苷、当药苷在滇龙胆根、茎、叶、花中均有分布;龙胆苦苷、獐牙菜苦苷在根部含量较高,分别为(35.88±5.89)mg/g和(1.35±0.33)mg/g;当药苷在花部含量最高,为(0.97±0.24)mg/g.(2)不同部位有效成分间具有显著(P<0.05)或极显著(P<0.01)的相关性.(3)相同海拔条件下,不同栽培方式下滇龙胆根部龙胆苦苷含量差异不显著(P>0.05),但茎部、叶部獐牙菜苦苷含量差异显著(P<0.05).     

滇龙胆GrHDR基因的克隆与表达分析

龙胆苦苷(gentiopicroside)是中药龙胆中的主要药效成分,属于萜类化合物的衍生物。1-羟基-2-甲基-2-(E)-丁烯基-4-二磷酸还原酶[1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase,HDR]是萜类物质合成途径中的关键酶。为探讨不同光照条件下滇龙胆HDR(GrHDR)基因的表达与龙胆苦苷含量之间的关系,该文以滇龙胆叶片cDNA为模板,采用PCR和TA克隆技术获得GrHDR基因序列,对该序列进行生物信息学分析和表达分析,并采用高效液相色谱法测定龙胆苦苷含量,对该基因表达与龙胆苦苷含量进行比较。结果表明:(1)GrHDR基因(GenBank登录号: KJ917165.1)全长1 398 bp,编码465个氨基酸,推定GrHDR蛋白是亲水且稳定的,相对分子质量是52 281.25 Da,理论等电点是5.32;(2)该蛋白属于LYTB蛋白家族,可能定位于叶绿体上,无信号肽,二级结构主要由α-螺旋(45.16%)、β-转角(6.24%)、无规卷曲(33.98%)、延伸链(14.62%)构成;(3)GrHDR蛋白序列与同属植物秦艽的HDR蛋白相似性最高(95.71%);(4)实时荧光定量PCR结果显示GrHDR基因在滇龙胆中的表达量为根 > 叶 > 茎,而在10%、30%、100%全光光照条件下各组织的表达量有很大差异;(5)高效液相色谱法结果显示,不同光照条件下龙胆苦苷含量一致,均为根 > 叶 > 茎,其中100%全光光照下,药用部位根中龙胆苦苷含量达到7.141%,约是30%、10%全光光照条件的两倍,但该结果与同一光照条件下GrHDR基因表达规律不完全一致。该研究为阐述HDR基因功能及其与龙胆苦苷含量的关系提供参考。     

农林复合系统滇龙胆茎叶化学计量特征研究

以农林复合系统种植的滇龙胆(Gentiana rigescens Franch.ex Hemsl.)为材料,采用高效液相色谱法建立不同栽培系统滇龙胆茎、叶的色谱指纹图谱,并测定其主要活性成分马钱苷酸、獐牙菜苦苷、龙胆苦苷和当药苷含量,研究不同栽培系统滇龙胆茎、叶化学计量特征。采用相关性分析、指纹图谱相似度分析、偏最小二乘判别分析(PLS-DA)、变量投影重要性准则(VIP)等方法进行化学数据分析。结果显示,滇龙胆主要活性成分马钱苷酸含量为(1.85±0.92)mg/g~(7.43±7.64)mg/g,獐牙菜苦苷含量为(1.03±0.17)mg/g~(1.58±0.50)mg/g,龙胆苦苷含量为(15.28±11.34)mg/g~(24.59±7.84)mg/g,当药苷含量为(4.10±1.64)mg/g~(31.67±22.70)mg/g,且叶片中4种活性成分的总含量高于茎;不同栽培系统中,与尼泊尔桤木间作的滇龙胆茎、叶活性成分总含量最高,而与核桃间作的滇龙胆茎、叶活性成分总含量最低。相关性分析显示,植株相同部位和不同部位间的环烯醚萜和裂环烯醚萜含量呈显著(P0.05)或极显著(P0.01)正相关。指纹图谱相似度分析表明,不同栽培系统滇龙胆茎指纹图谱相似度介于0.989~0.992之间、叶指纹图谱相似度为0.988~0.996,相同部位样品化学成分种类相似。PLS-DA分析结果表明,茎和叶片整体化学计量特征具有明显差异;单作及林药间作的样品被区分为不同类群,不同间作模式下滇龙胆茎、叶化学成分具显著差异,叶片高效液相色谱指纹图谱可用于区分不同栽培系统滇龙胆样品。本研究结果可为农林复合系统滇龙胆有效成分含量研究及滇龙胆资源的合理开发利用提供科学依据。     

滇龙胆中萜类物质积累的动态变化

滇龙胆（Gentiana rigescens）为传统中药材。采用单因素方差分析比较不同生长季节滇龙胆的根部及茎叶部位龙胆苦苷、獐牙菜苦苷及当药苷含量的动态变化。结果表明,在不同生长季节,滇龙胆的根部与茎叶部位3种有效成分的含量具明显差异。龙胆苦苷主要积累于根部;獐牙菜苦苷、当药苷主要积累于茎叶部位。相关性分析表明,龙胆苦苷含量受气候因子的影响,月平均温度与茎叶部位龙胆苦苷含量呈极显著负相关（R=-0.57,P〈0.01）,月降水量与根部、茎叶部位龙胆苦苷含量呈显著（R=-0.48,P〈0.05）或极显著（R=-0.74,P〈0.01）负相关;根中当药苷含量与獐牙菜苦苷含量呈极显著正相关（R=0.62,P〈0.01）,茎叶部位獐牙菜苦苷含量与当药苷含量呈显著正相关（R=0.38,P〈0.05）。研究结果表明,滇龙胆中龙胆苦苷含量受降水量和温度的影响;龙胆苦苷、獐牙菜苦苷及当药苷的含量变动具相关性;9-11月较适宜滇龙胆药材的采收。     

条叶龙胆根培养物中龙胆苦苷的产生和含量

条对龙胆愈伤组织中龙胆苦苷含量较低,根的分化可以提高龙胆苦苷的含量。培养的离休根中龙胆苦苷的含量则更高。随着继代次数的增加,愈伤组织中龙胆苦苷含量下降快,而分化根的愈伤组织和离体培养根中龙胆苦苷含量下降较慢。在愈伤组织的液体培养中,1/2MS培养基附加低浓度的植物激素,龙胆苦苷含量有所提高。植物激素配比促进愈伤组织中分化根的形成,也促进龙胆苦苷的产生;但促进愈伤组织的生长时,不利于龙胆苦苷产生。在离体根培养中,添加 1mg/L的金雀花碱,促进龙胆苦苷的产生,含量可达1.48％,明显高于愈伤组织(0.52％)和分化根的愈伤组织(0.65％),也高于试管苗根的龙胆苦苷含量(1.01％)。     

粗茎秦艽不同部位龙胆苦甙含量的分析

采用高压液相法分析了栽培粗茎秦艽不同部位的有效药用成分龙胆苦甙(gentiopicrin)的含量,并与野生麻花秦艽、达乌里秦艽龙胆苦甙的含量相比较。结果表明粗茎秦艽根、茎、叶均具有较高的开发利用价值,为合理开发利用粗茎秦艽提供了科学依据。     

藏药提宗龙胆和线叶龙胆花中四种苦苷类成分的HPLC测定

应用HPLC分析方法同时测定藏药提宗龙胆和线叶龙胆两种植物花中落干酸、獐牙菜苦苷、龙胆苦苷、獐牙菜苷4种苦苷类成分的含量.采用Econosphere C18色谱柱(250×4.6 mm,5 μm),以甲醇-0.5%乙酸为流动相进行梯度洗脱,流速为1.0 mL/min;检测波长为245 nm.结果表明,除提宗龙胆中未检出獐牙菜苦苷外,其它成分均在两种植物中存在,但含量存在一定的差异.     

响应面法优化线叶龙胆中龙胆苦苷提取工艺

采用响应面法优化乙醇热回流提取线叶龙胆中龙胆苦苷的提取工艺。在单因素试验基础上,选择乙醇浓度、料液比、提取时间为自变量,以龙胆苦苷提取率为响应值,进行Box-Behnken中心组合实验设计,采用响应面法(RSM)评估其对龙胆苦苷提取率的影响。在乙醇浓度71%,液料比34:1,提取时间83min时,提取率达4.52%。     

High-frequency embryogenesis and regeneration of plants with high content of gentiopicroside from the Chinese medicinal plant Gentiana straminea Maxim.

Gentiana straminea Maxim. (MahuaQinjiao) is an important Chinese medicinal herb that is rich in secoiridoids. To increase the production of this plant, we have developed an efficient tissue culture system for high-frequency plant regeneration. We have also obtained plants with high secoiridoid monomer production through tissue culture and somatic embryogenesis. We have found that the MB medium is better than the B5 medium for callus induction. Calluses induced from immature seeds are superior to those from hypocotyls or young leaves in regeneration via somatic embryogenesis. We have also demonstrated that 2,4-dichlorophenoxyacetic acid is efficient for both callus induction and embryogenesis, indole-3-acetic acid is suitable for embryogenic callus proliferation, and N ⁶-(benzyl)-adenine promotes both embryo development and the accumulation of gentiopicroside in the cultures. Regenerated plants have been selected for high gentiopicroside content. One plant contains 5.82% of gentiopicroside, which is higher than the control plants (1.20–3.73%). The regenerated plants are genetically more stable than the calluses based on both cytological and random amplified polymorphic DNA analyses.     

太子参细胞悬浮培养及其皂苷含量分析

以太子参的幼叶为外植体,诱导培养获得太子参愈伤组织,并通过细胞悬浮培养获取皂苷.结果表明：用MS＋BA 0.2 mg L^-1＋2,4-D 1.0 mg L^-1＋KT 1.0 mgL^-1液体培养基可获得大量繁殖速度快、生长均匀一致的悬浮细胞.由细胞悬浮培养获得的太子参皂苷的HPLC色谱峰值与常规种植及组培苗的相同,但纯度较好.细胞悬浮培养约30 d时,每克干重细胞的培养液内可提取总皂苷量为2.13-2.92 mg,略低于大田常规种植所收获的每克干重太子参块根内的总皂苷含量（3.6-4.3 mg）,与组培苗收获的太子参块根内的总皂苷含量相近.     

霍山石斛组培苗移栽后微观结构与微区元素的变化研究

采用扫描电镜及X射线能谱仪技术,研究霍山石斛组培苗移栽后各器官组织的微观结构和所含元素的变化,以了解霍山石斛组培苗生长发育以及药效成分的状况。结果表明:(1)霍山石斛组培苗移栽2个月后,根增粗近1倍,具有根被,且根被细胞壁的网络状增厚更加明显,表皮、皮层、中柱发育分化更加完善,Mg、Si、Cl、S、K、Ca元素含量提高,其中Ca增长了2.76倍。(2)茎表面纵向凹陷增多,内部结构致密,维管束发育较为完善,细胞内充满物质,Mg、Si、Cl、S、K、Ca、Fe、Cu、Zn元素含量增多,尤其是K和Fe分别增长了3.25倍和4.61倍。(3)叶增厚,具有了角质层,气孔形状更加饱满,叶肉细胞内含物丰富,Mg、Si、Cl、S、K、Ca、Fe、Mn、Zn的含量显著上升,其中K升高了17倍。(4)与根、叶相比,茎中所富集的元素种类最多。研究认为,霍山石斛试管苗移栽后,生长发育趋向完善,各项功能增强,细胞内含物质增多,元素成分丰富并且含量提高,体现出对移栽环境的良好适应性。     

铁皮石斛组培苗移栽驯化过程中叶片光合特性、 超微结构及根系活力的变化

研究了铁皮石斛(Dendrobium candicum Wall.ex Lindl.)组培苗在移栽驯化(即试管苗移栽大田)过程中,移栽0、1、2、4d和20d(已移栽成活)时叶片光合特征参数、叶绿素荧光特性及叶绿体超微结构,光合色素含量、可溶性糖含量、根系活力和硝酸还原酶活性等生理特征的变化,以阐明铁皮石斛组培苗在移栽驯化过程中发生的适应性变化。结果表明:铁皮石斛幼苗在移栽驯化过程中叶片表观量子效率、最大净光合速率、羧化速率以及PSⅡ的最大光化学效率和表观光合电子传递速率均在移栽驯化过程中不断升高并于20d时达到最大;而暗呼吸速率和叶绿素b的含量在移栽20d时显著降低。驯化过程中叶片光合机构遭到破坏,但移栽20d时叶绿体内淀粉粒和嗜饿小体体积变大,片层结构清晰、完整且有序。根系活力和硝酸还原酶活性均在移栽20d时显著增大。铁皮石斛组培苗在移栽驯化过程中叶片光合特性、超微结构及根系活力都发生了一定的适应性变化,随着移栽时间的延长,石斛叶片的光合效率明显提高,并有大量的光合产物积累;同时根系吸收水分、养分的能力及抗逆能力都明显增强,到移栽20d时组培苗已完全适应大田环境。     

不同培育方式对橡胶树实生苗生理指标、养分及长势的影响

橡胶树(Heveabrasiliensis)种子催芽生长一般使用沙床培育,沙子是不可再生资源,为了选择一种适合橡胶树种子培育方式来替代对沙子的依赖,该研究通过水培、悬空培育和传统的沙培比较橡胶树实生苗第1蓬叶稳定时,苗木的生长势、生理指标及养分含量。结果表明,水培实生苗地上部株高、茎粗、叶面积的长势最佳,壮苗指数和生物量的含量最高,但其根太长,根相对较细。水培的叶、茎、根的可溶性糖、丙二醛、游离脯氨酸、超氧化物歧化酶的含量均较低;水培和悬空培育的叶片和茎的叶绿素、类胡萝卜素及根系活力的含量没有显著性差异,均高于沙培。水培的叶、茎、根中的氮和磷含量最低,沙培的最高;而水培实生苗根和茎中钾的含量较高,叶片中含量与悬空培育、沙培均没有显著性差异;悬空培育在叶、茎、根中钾的含量最低。水培促进了苗木的生长,降低干旱胁迫,提高养分利用率,但后续还需调控根系,建设良好根团。悬空培育的苗木长势较弱,还需进一步完善方法。     

HPLC Determination of Contents of Four Active Constituents in Tibetan Medicine Gentiana straminea ( Gentianaceae) During Different Growing Period

Contents of four iridoid glycosides viz. gentiopicroside, longanic acid, swertiamarin and sweroside of wild and cultivated Gentiana straminea roots gathered in different seasons were analyzed by means of HPLC determination. The results indicated that contents of the four iridoid glycosides varied greatly with different seasons between wild and cultivated Gentiana straminea. And according to the criterion of pharmacopoeia of China, after cultivation content of gentiopicroside in Gentiana str aminea root had accorded with the regulation and can preliminarily replaced the wild species to use as a plant medicine. The mechanism causing the content changes of the four iridoid glycosides may be due to the integrated effects of many factors, such as the ecological factors, the genetic factors.     

不同生长季节下藏药麻花秦艽活性成分含量研究

采用高效液相色谱法测定了野生与栽培藏药麻花秦艽（Gentiana straminea）根中龙胆苦苷、落干酸、獐牙菜苦苷和獐牙菜苷四种环烯醚萜苷类化学成分的含量及其在不同生长季节的变化趋势。结果表明,4种环烯醚萜苷类成分的含量随植物的生长季节而波动,其活性成分含量在野生种与栽培种之间发生了一定的差异。但是,栽培根中的龙胆苦甙已达到药典的标准,可供药用。     

Composition and content of glucosinolates in developing Arabidopsis thaliana

The glucosinolate composition and content in various tissues of Arabidopsis thaliana (L.) Heynh. ecotype Columbia during development from seeds to bolting plants were determined in detail by high-performance liquid chromatography. Comparison of the glucosinolate profiles of leaves, roots and stems from mature plants with those of green siliques and mature seeds indicated that a majority of the seed glucosinolates were synthesized de novo in the silique. A comparison of the glucosinolate profile of mature seeds with that of cotyledons indicated that a major part of seed glucosinolates was retained in the cotyledons. Turnover of glucosinolates was studied by germination of seeds containing radiolabelled p-hydroxybenzylglucosinolate (p-OHBG). Approximately 70% of the content of [14C]p-OHBG in the seeds was detected in seedlings at the cotyledon stage and [14C]p-OHBG was barely detectable in young plants with rosettes of six to eight leaves. The turn-over of p-OHBG was found to coincide with the expression of the glucosinolate-degrading enzyme myrosinase, which was detectable at very low levels in seedlings at the cotyledon stage, but which dramatically increased in leaves from plants at later developmental stages. This indicates that there is a continuous turnover of glucosinolates during development and not only upon tissue disruption.     

Propagation of <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> (Pall) from hairy root explants via indirect somatic embryogenesis and gentiopicroside content in obtained plants

An effective protocol for plant regeneration from hairy root (HR) via indirect somatic embryogenesis was established in medicinal plant Gentiana macrophylla, a perennial herb in Gentianaceae. On the MS medium containing 0.5–2.5 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D plus benzylaminopurine (BAP), all the HR explants produced embryogenic calli (ECs). After transfer to plant growth regulator (PGR)-free MS medium, up to 94% of the ECs produced somatic embryos (SEs) of various stages, including cotyledonary SEs. When the calli with cotyledonary SEs were transferred to PGR-free MS medium, the cotyledonary SEs on the calli developed into plantlets (1–12 ones per callus). The cotyledonary SEs showed two types: solitary and fasciculate. The former developed into single plantlets and the latter into fasciculate ones. After transplantation into soil, a half of the plantlets survived, and one of the survivors flowered without fruiting. Morphologically, about 30% plantlets appeared similar to the wild type (WT)-plants, and 70% of them displayed wrinkled dark green leaves with relatively small and dense stomata, long and thick main root with dense lateral roots. The biomass of roots and leaves of the plantlets increased by five- and one-fold, respectively, and the content of gentiopicroside of their roots raised by 72.4%, in comparison with WT-plants. Polymerase chain reaction revealed that the rolC gene integrated into HR genome still existed in the regenerated plants. This study offers us an effective method and material for producing gentiopicroside or other medicinal compounds.